Summary/Abstract for R21 Pyridine nucleotides are redox coenzymes that are essential for bioenergetics, metabolism and DNA damage responses. For decades, autofluorescence from reduced pyridine nucleotides (i.e., NADH and NADPH or NAD(P)H) was used to study redox in the nucleus, cytoplasm and mitochondria, while the endoplasmic reticulum (ER) NAD(P)H pool was assumed to be insignificant. This assumption has now been overturned by our recent brain slice two-photon NAD(P)H imaging experiments that demonstrated that much of somatic NAD(P)H in dopamine neurons is in the ER and coupled to mitochondrial function. We also discovered that an amphetamine acts via dopamine vesicles to rapidly reduce NAD(P)H, thus potentially reducing compensation for redox stress. Therefore, our studies have revealed new NAD(P)H organelle distribution and redox coupling, as well as NAD(P)H regulation by an important abused drug. Here we use fluorescence lifetime microscopy (FLIM) and newly generated ER- targeted indicators for individual pyridine nucleotides and their redox ratios (e.g. NAD+/NADH) to determine the identity and location of pyridine nucleotides involved in the amphetamine response and ER-mitochondria NAD(P)H coupling. Then the roles of shuttles and ER-mitochondria proximity in interorganelle redox coupling will be determined. Finally, the ER NAD(P)H pool will be perturbed to test the novel hypothesis that the ER buffers pyridine nucleotide redox in other organelles. This study will yield fundamental insights into the native and pharmacological control of pyridine nucleotides in organelles and guide future development of therapies for reducing dopamine neuron redox stress associated with Parkinson?s disease and amphetamine abuse.